La perméabilité vasculaire est une caractéristique cruciale de l’angiogenèse. Les acteurs principaux sont les cellules endothéliales qui la régulent en réponse à divers facteurs perméabilisant, tels que le « Vascular Endothelial Growth Factor » (VEGF). Dans le contexte pathologique du cancer, les cellules tumorales produisent de grandes quantités de VEGF qui stimulent la perméabilité, ce qui leur permet d’infiltrer le réseau vasculaire. Il est connu que la tyrosine kinase Src contrôle cette modulation de la perméabilité. Puisque notre laboratoire a préalablement démontré que la phosphatase de type récepteur (PTP) DEP-1 est impliquée dans l’activation de Src en réponse au VEGF, nous avons émis l'hypothèse que DEP-1 pourrait aussi jouer un rôle dans la perméabilité des cellules endothéliales. Grâce à des expériences de transfections d’ARN interférant, nous démontrons que DEP-1 est important pour la régulation de la phosphorylation de la VE-Cadhérine, un médiateur critique de la perméabilité. L’impact de DEP-1 sur la dissociation de jonctions intercellulaires est également démontré par microscopie à immunofluorescence de cellules endothéliales. DEP-1 est également nécessaire à l’augmentation de la perméabilité induite par VEGF in vitro. Deux résidus tyrosine retrouvés dans la queue carboxy-terminale de DEP-1 sont essentiels à l’activation de Src en réponse au VEGF. Suite à la transfection d’un plasmide encodant DEP-1 muté pour ces deux résidus, nous démontrons aussi leur implication dans la régulation de la perméabilité in vitro par DEP-1. Ces travaux permettent ainsi d’approfondir nos connaissances sur un nouveau régulateur potentiel de la perméabilité vasculaire. / Endothelial cell permeability is a crucial step of angiogenesis. The main actors behind permeability are endothelial cells who accomplish this in response to permeabilizing factors, most notably Vascular Endothelial Growth Factor (VEGF). In a pathological context, migrating tumor cells produce great quantities of VEGF that stimulate an increase of vascular permeability, which allows them to intravasate into the vasculature. Src has been shown to mediate this process. Our laboratory has previously shown that the protein tyrosine phosphatase DEP-1 is involved in the regulation of VEGF-dependant activation of Src. These data thus suggested that DEP-1 might play a role in endothelial cell permeability. Here, we show through siRNA experiments that DEP-1 is important for the regulatory phosphorylation of VE-Cadherin which is critical for the induction of permeability. The impact of DEP-1 on intercellular junction dissociation is also demonstrated through immunofluorescence microscopy of endothelial cells. We further show that DEP-1 is absolutely required for the VEGF-dependent increase of permeability as illustrated by in vitro permeability assay on siRNA-transfected endothelial cells. Finally, we show that tyrosine residues in DEP-1’s carboxy-terminal tail, which are crucial for mediating Src activity in response to VEGF, are implicated in VEGF-dependant increase in permeability by transfecting plasmids coding for DEP-1 mutants of these tyrosine residues. These findings shed light on a novel potential key regulator of in vivo permeability.

Zugl. Kurzfassung von: München, Univ., Diss., 2006

A tendência atual, imposta pela competitividade do mercado, exige bovinos mais eficientes e adequados ao sistema de produção, visando atender a demanda pela qualidade de carne. Em função disso, o melhoramento genético vem sendo utilizado, de forma a incrementar a eficiência dos rebanhos, proporcionando assim, maior rentabilidade ao sistema. Assim, o presente estudo foi conduzido com o propósito de estudar a variabilidade da progênie de touros submetidos à avaliação genética, através da análise das Diferenças Esperadas na Progênie (DEP) dos filhos de 359 reprodutores. As DEP das progênies foram estimadas por procedimentos REML e as avaliações de touros por metodologias alternativas, foram realizadas com o auxílio do software SAS. Foram utilizados 45.014 dados de peso à desmama (PD), 45.014 de peso ao sobreano (PS) e 45.014 de ganho de peso da desmama ao sobreano (GP) de bovinos de corte da raça Nelore, criados em uma fazenda em região tropical do Brasil. As DEPs dos touros foram estimadas de acordo com três métodos: uso da média aritmética, média harmônica acrescida da média da característica estudada e média harmônica simples. Os resultados sugerem que o uso da média harmônica na estimação de DEPs com o objetivo de ajustar o valor genético aditivo de um touro para a dispersão dos valores genéticos de suas progênies não trouxe diferenças importantes em relação à estimativa pela média aritmética, na população estudada. No entanto, dadas as implicações e importância de novas metodologias, são necessários maiores estudos em busca de se encontrar uma metodologia que permita maior uniformidade das progênies de touros. / The strong competition in beef market imposed an actual trend of searching for quality in beef production systems, trying to attend the consumers\' demand. Due to the necessity of increasing productivity, animal breeding programs have been utilized to increase cattle efficiency, offering more profitability to the production system. The present study was carried out to contribute to knowledge about sires\' progeny variability, using Expected Progeny Difference (EPD) from calves of 359 sires. EPD\'s were estimated by REML procedures. The prediction od bull\'s EPD\'s were performed by software SAS, using 45,014 data of weaning weight (PD), 45,014 of post-weaning weight (PS) and 45,014 of weight gain post-weaning (GP) from Nellore beef cattle, raised in a tropical area of Brazil. Those data were analyzed according with three methods: application of arithmetic mean, harmonic mean of EPD added by trait mean and only harmonic mean. Results suggested that estimation of EPDs using harmonic mean on condition that adjust sire\'s additive genetic value to dispersion form its calves\' genetic values did not make difference to the prediction trough arithmetic mean in the population studied. Due to the implications and importance of alternative methods to adjust bull\'s EPDs to variability of progeny, further studies are necessary to search for better progeny\'s uniformity.

No description available.

Today the medical field is struggling to decrease bacteria biofilm formation which leads to infection. Also, biomedical devices sterilization has not changed over a long period of time which has resulted in high costs for hospitals healthcare managements. The objective of this project is to investigate electro-dynamic effects by surface energy manipulation as potential methods for preventing bacteria biofilm growing on medical devices. Based on electrokinetic environments two different methods were tested: rejection bacteria dielectrophoretic forces feasibility by numerical simulations; and electrowetting-on -dielectric by the fabrication of golden interdigitated electrodes on silicon glass substrates covered by a Teflon layer. In the first experiment, numerical simulations of gold electrodes in buffer solution and frequencies were carried out to determine the forces required to reject bacteria. In the second experiment, interdigitated gold electrodes coated with a dielectric Teflon layer, were characterized in terms of breakdown voltage, dielectric adhesion and contact angle in terms of applied voltage. Finally the effect of EWOD on bacterial adhesion was tested. The project resulted in promising simulation results for bacteria rejection using dielectrophoresis due to the wide range of frequency that rejects the modelled bacteria. However, practical experiments such as electrowetting-on-dielectric must verify this at incubation times larger than 24 hours in spite of the Teflon non-adhesive properties. / <p>opponent Alex Grossm ann Colin</p> / VRI

Pathogens in drinking water are the cause of over 1.5 million deaths around the world every year, mostly in developing countries. Practical, cheap, and effective tools for detection of these pathogens are critical to advance public health in many areas around the globe. Micro electro-mechanical systems (MEMS) are miniaturized structures that can be used for a variety of purposes, including, but not limited to, small scale sensors. Therefore, MEMS can be used in place of expensive laboratory equipment and offer a cheap and practical tool for pathogen detection. The presented work's research objective is to introduce a new technique called embedded passivated-electrode insulator-based dielectrophoresis (EπDEP) for preconcentration, separation, or enrichment of bioparticles, including living cells. This new method combines traditional electrode-based DEP and insulator-based DEP with the objective of enhancing the electric field strength and capture efficiency within the microfluidic channel while alleviating direct contact between the electrode and the fluid. The EπDEP chip contains embedded electrodes within the microfluidic channel covered by a thin passivation layer of only 4 μm. The channel was designed with two nonaligned vertical columns of insulated microposts (200 μm diameter, 50 μm spacing) located between the electrodes (600 μm wide, 600 μm horizontal spacing) to generate the nonuniform electric field lines to concentrate cells while maintaining steady flow in the channel. The performance of the chip was demonstrated using Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial pathogens in aqueous media. Trapping efficiencies of 100% were obtained for both pathogens at an applied AC voltage of 50 V peak-to-peak and flow rates as high as 10 uL/min. / Master of Science

The focus of this research is the isolation of waterborne pathogens which are one of the grand challenges to human health, costing the lives of about 2.5 million people worldwide each year. The aim was to develop new microfluidic techniques for selectively concentrating and detecting waterborne pathogens. Detection of microbes in water can greatly help reduce deaths; however, analytical instruments cannot readily detect them due to the extreme dilution of these microbes, and hence, require significant sample concentration. Current methods are expensive and either require days to process or are not sufficiently robust for water monitoring. Microfluidic chips based on insulator-based dielectrophoresis (iDEP) provide a promising solution to these problems and have been previously used to selectively concentrate biological particle such as bacteria. The microfluidic devices in this work were created with a 3D mircofabrication technique, which we also developed as part of this project. The core process of the technique is the etching of 3D structures in silicon with a single plasma etch utilizing an effect known as reactive ion etch lag (RIE lag). Using this unique process, 3D devices are fabricated in both silicon and the polymer polydimenthylsiloxane (PDMS). Using both numerical modeling and experimental results, we show how these 3D structures enhance the performance of the dielectrophoretic devices. The main findings indicate that 3D structures can help reduce Joule heating in the devices and lower the applied voltage necessary to operate the devices. Additionally, within this work, we develop a new dielectrophoresis technique called off-chip passivated-electrode, insulator-based dielectrophoresis microchip (O"DEP). This technique combines the sensitivity of electrode-based dielectrophoresis (eDEP) with the high-throughput and inexpensive device characteristics of insulator-based dielectrophoresis. The result is a cartridge based system which is accessible, economical, high-performance, and high-throughput technologies allowing timely detection of pathogenic bacteria. / Ph. D.

This work was focused on the characterization of microparticles with particular emphasis on waterborne pathogens which pose a great health risk to human lives. The goal of this study was to develop microfluidic systems for enhanced characterization and isolation of bioparticles. Insulator-based dielectrophoresis (iDEP) is a promising technique for analyzing, characterizing and isolation of microparticles based on their electrical properties. By employing insulator-based constrictions within the microchannel in combination with microelectrodes within the vicinity of the electrodes, dielectrophoretic performance is enhanced. In this study, three dimensional insulator-based dielectrophoresis devices are fabricated using our in-house developed 3D micromachining technique. This technology combines the benefits of electrode-based DEP, insulator-based DEP, and three dimensional insulating features with the goal of improving trapping efficiency of biological species at low applied signals and fostering wide frequency range operation of the microfluidic device. The dielectric properties of bacteria as well as submicron polystyrene beads are discussed and the impact of these results on the future development of iDEP microfluidic systems is explored. / Master of Science

L’implication des protéines tyrosines phosphatases (PTPs) dans la régulation de la signalisation et la médiation des fonctions cellulaires a été bien établie dans les dernières années. Cependant, les mécanismes moléculaires par lesquels les PTPs régulent les processus fondamentaux tels que l’angiogenèse demeurent méconnus. Il a été rapporté que l’expression de la PTP DEP-1 (Density-enhanced phosphatase 1) augmente avec la densité cellulaire et corrèle avec la déphosphorylation du récepteur VEGFR2. Cette déphosphorylation contribue à l’inhibition de contact dans les cellules endothéliales à confluence et diminue l’activité du VEGFR2 en déphosphorylant spécifiquement ses résidus catalytiques Y1054/1059. De plus, la plupart des voies de signalisation en aval du VEGFR2 sont diminuées sauf la voie Src-Gab1-AKT. DEP-1 déphosphoryle la Y529 de Src et contribue à la promotion de la survie dans les cellules endothéliales. L’objectif de cette thèse est de mieux définir le rôle de DEP-1 dans la régulation de l’activité de Src et les réponses biologiques dans les cellules endothéliales. Nous avons identifié les résidus Y1311 et Y1320 dans la queue C-terminale de DEP-1 comme sites majeurs de phosphorylation en réponse au VEGF. La phosphorylation de ces résidus est requise pour l’activation de Src et médie le remodelage des jonctions cellules-cellules dépendantes de Src. Ce remodelage induit la perméabilité, l’invasion et la formation de capillaires en réponse au VEGF. Nos résultats démontrent que la phosphorylation de DEP-1 sur résidu tyrosine est requise pour diriger la spécificité de DEP-1 vers son substrat Src. Les travaux révèlent pour la première fois un rôle positif de DEP-1 sur l’induction du programme angiogénique des cellules endothéliales. En plus de la phosphorylation sur tyrosine, DEP-1 est constitutivement phosphorylé sur la thréonine 1318 situé à proximité de la Y1320 en C-terminal. Cette localisation de la T1318 suggère que ce résidu pourrait être impliqué dans la régulation de la Y1320. En effet, nous avons observé que la T1318 de DEP-1 est phosphorylée potentiellement par CK2, et que cette phosphorylation régule la phosphorylation de DEP-1 sur tyrosine et sa capacité de lier et d’activer Src. En accord avec ces résultats, nos travaux révèlent que la surexpression du mutant DEP-1 T1318A diminue le remodelage des jonctions cellules-cellules et par conséquent la perméabilité. Nos résultats suggèrent donc que la T1318 de DEP-1 constitue un nouveau mécanisme de contrôle de la phosphorylation sur tyrosine et que ceci résulte en l’activation de Src et l’induction des fonctions biologiques des cellules endothéliales en réponse au VEGF. Suite à ces travaux dans les cellules endothéliales qui démontrent un rôle positif de DEP-1 dans la médiation des réponses angiogéniques, nous avons voulu approfondir nos connaissances sur l’implication potentielle de DEP-1 dans les cellules cancéreuses où l’activité de Src est requise pour la progression tumorale. Malgré le rôle connu de DEP-1 comme suppresseur tumoral dans différents types de cancer, nous avons émis l’hypothèse que DEP-1 pourrait promouvoir les fonctions biologiques dépendantes de Src telles que la migration et l’invasion dans les cellules cancéreuses. Ainsi, nous avons observé que l’expression de DEP-1 est plus élevée dans les lignées basales de cancer du sein qui sont plus invasives comparativement aux lignées luminales peu invasives. Dans les lignées basales, DEP-1 active Src, médie la motilité cellulaire dépendante de Src et régule la localisation des protéines impliquées dans l’organisation du cytosquelette. L’analyse d’un micro-étalage de tissu a révélé que l’expression de DEP-1 est associée avec une réduction tendencielle de survie des patients. Nos résultats proposent donc, un rôle de promoteur tumoral pour DEP-1 dans la progression du cancer du sein. Les travaux présentés dans cette thèse démontrent pour la première fois que DEP-1 peut agir comme promoteur des réponses angiogéniques et du phénotype pro-invasif des lignées basales du cancer du sein probablement du à sa capacité d’activer Src. Nos résultats suggèrent ainsi que l’expression de DEP-1 pourrait contribuer à la progression tumorale et la formation de métastases. Ces découvertes laissent donc entrevoir que DEP-1 représente une nouvelle cible thérapeutique potentielle pour contrer l’angiogenèse et le développement du cancer. / The implication of protein tyrosine phosphatases (PTPs) in the regulation of cell signalling events and the mediation of cellular functions in response to growth factors such as VEGF has been well-established in the last years. Nonetheless, molecular mechanisms by which PTPs regulate fundamental processes such as angiogenesis are not well-characterized. Expression of the PTP DEP-1 (Density-enhanced phosphatase 1) was reported to increase with cell density and was associated with VEGFR2 dephosphorylation contributing to cell contact inhibition in confluent endothelial cells. We previously demonstrated that DEP-1 attenuates VEGFR2 activity by dephosphorylation of its Y1054/1059 leading to decreased activation of major signalling pathways downstream of VEGFR2 with exception of the Src-Gab1-AKT pathway. Increasing Src activity due to DEP-1-mediated dephosphorylation of its Y529 promotes endothelial cell survival. The objective of this thesis was to gain a better understanding of the role of DEP-1 in the regulation of the Src activity and of biological responses in endothelial cells. We identified tyrosine Y1311 and Y1320 in the C-terminal tail of DEP-1 as major phosphorylation sites in response to VEGF. These residues are required for Src activation and mediate the Src-dependent remodelling of endothelial cell junctions inducing permeability, invasion and capillary formation upon VEGF stimulation. We showed that VEGF-induced DEP-1 tyrosine phosphorylation directs DEP-1 specificity towards its substrate Src. Our results thus highlighted for the first time the promoting role of DEP-1 on the angiogenic program in endothelial cells. In addition to tyrosine phosphorylation, DEP-1 is constitutively phosphorylated on a threonine residue (T1318) proximal to Y1320 in its C-terminal tail suggesting it might be involved in the regulation of Y1320. Indeed, we found that DEP-1 T1318 is phosphorylated, potentially by CK2, and regulates the tyrosine phosphorylation of DEP-1 and its ability to bind to and activate Src. Consistent with this, remodelling of endothelial cell junctions and permeability are impaired in endothelial cells expressing the DEP-1 T1318 mutant. Thus, DEP-1 phosphorylation on T1318 displays a regulatory control over DEP-1 tyrosine phosphorylation and subsequently Src activation and endothelial cell functions in response to VEGF. Our results demonstrating that DEP-1 promotes angiogenic cell responses in endothelial cells, prompted us to consider a possible involvement of DEP-1 in cancer cells, where Src activation has been linked to cancer progression. Thus, although, DEP-1 is believed to act as a tumour suppressor in different cancer types, we hypothesized that it might also promote Src-dependent functions such as migration and invasion in cancer cells. Interestingly, we found that DEP-1 is higher expressed in more invasive basal-like breast cancer cells than in luminal-like cell lines. Moreover, DEP-1 is implicated in the regulation of Src activity, Src-mediated cell motility and appropriate localization of proteins mediating cytoskeletal organization in basal-like breast cancer cell lines. To further support these results, analysis of a breast cancer tissue microarray revealed that DEP-1 expression is associated with a tendency towards reduced overall survival. Thus, our results provide first evidence for a tumour-promoting role of DEP-1 in breast cancer. Altogether, the work performed in the context of this thesis revealed that DEP-1 can similarly behave as a promoter of the angiogenic response and of the pro-invasive phenotype in basal-like breast cancer cell lines, most likely due to its ability to activate Src. This suggests for the first time that DEP-1 expression could contribute to tumour progression and the formation of metastases, and as such, represent a potential new target for anti-angiogenic and anti-cancer therapy.

Os &eacute;steres de ftalato (PAEs) s&atilde;o compostos produzidos em grandes quantidades, amplamente utilizados industrialmente como agentes plastificantes. Seus res&iacute;duos s&atilde;o lixiviados pela &aacute;gua tornando-se poluentes org&acirc;nicos persistentes (POPs) no meio ambiente aquoso, al&eacute;m de apresentar caracter&iacute;sticas de interfer&ecirc;ncia end&oacute;crina. O dietil ftalato (DEP) &eacute; frequentemente encontrado nas amostras ambientais, pois possui elevada solubilidade na &aacute;gua e pode ser gerado durante a degrada&ccedil;&atilde;o de outros PAEs. Assim, este trabalho teve como objetivo a degrada&ccedil;&atilde;o do dietil ftalato em meio aquoso por m&eacute;todo eletroqu&iacute;mico utilizando um &acirc;nodo dimensionalmente est&aacute;vel (ADE) comercial representado como Ti/Ru0,3Ti0,7O2 em uma c&eacute;lula do tipo filtro-prensa. As eletr&oacute;lises foram de 120 minutos contendo uma concentra&ccedil;&atilde;o inicial de 100,3 mg L-1 de DEP, pH inicial igual a 3, a temperatura em 25 &deg;C e vaz&atilde;o em 250 mL min-1. Os experimentos foram feitos utilizando planejamento fatorial do tipo 32 com duas r&eacute;plicas no ponto central, apresentando como vari&aacute;veis independentes a densidade de corrente (10, 25 e 40 mA cm-2) e o logaritmo em base 10 da forca i&ocirc;nica do eletr&oacute;lito suporte, NaCl e Na2SO4 (&micro; = 0,05, 0,15 e 0,5 mol L-1), com o intuito de estudar o efeito da densidade de corrente, concentra&ccedil;&atilde;o e natureza do eletr&oacute;lito para determinar a melhor condi&ccedil;&atilde;o de degrada&ccedil;&atilde;o do dietil ftalato. O monitoramento da concentra&ccedil;&atilde;o do DEP foi feito com cromatografia l&iacute;quida de alta efici&ecirc;ncia (CLAE) e a mineraliza&ccedil;&atilde;o foi acompanhada pelas an&aacute;lises de carbono org&acirc;nico total (COT). Foram obtidas maiores porcentagens de remo&ccedil;&atilde;o e mineraliza&ccedil;&atilde;o com uso das maiores densidades de corrente e na presen&ccedil;a de altas concentra&ccedil;&otilde;es de NaCl em compara&ccedil;&atilde;o com Na2SO4. Dessa maneira, se obteve remo&ccedil;&atilde;o de 63,2 % e mineraliza&ccedil;&atilde;o de 63,9 % em solu&ccedil;&atilde;o 0,5 mol L-1 NaCl e densidade de corrente de 40 mA cm-2, enquanto que para Na2SO4 (&micro; = 0,5 mol L-1) e 40 mA cm-2 foi removido 51,3 % e mineralizado 53,0 % de DEP. O mecanismo de degrada&ccedil;&atilde;o de DEP foi determinado em meio de NaCl e Na2SO4, atrav&eacute;s de CLAE-MS nas condi&ccedil;&otilde;es citadas anteriormente, identificando-se os &iacute;ons moleculares de m/z 149 e 177 em ambos eletr&oacute;litos, correspondentes ao anidrido ft&aacute;lico protonado e ao aduto do anidrido ft&aacute;lico com C(2)H(5)(+) respectivamente, &iacute;ons caracter&iacute;sticos da fragmenta&ccedil;&atilde;o do DEP, al&eacute;m do &iacute;on m/z 239 em Na2SO4 correspondente ao dietil 3-hidroxiftalato. A degrada&ccedil;&atilde;o do DEP acontece atrav&eacute;s da cadeia alif&aacute;tica. / Phthalate esters (PAEs) are compounds produced in large amounts industrially widely used as plasticizers. Their waste are leached by water becoming persistent organic pollutants (POPs) in the aqueous environment, besides having endocrine disrupting characteristics. Diethyl phthalate (DEP) is frequently found in environmental samples, it has high solubility in water and can be generated during the degradation of other PAEs. This work studied the degradation of diethyl phthalate in aqueous media by electrochemical method using a commercial dimensionally stable anode (ADE) represented as Ti/Ru0,3Ti0,7O2 in a filter-press flow cell. The electrolysis were carried out during 120 minutes containing an initial concentration of 100.3 mg L-1 DEP, initial pH = 3, temperature at 25 &deg;C and flow rate of 250 ml min-1. The experiments were performed using factorial design 32 with two replicas at the midpoint, with as independent variables the current density (10, 25 and 40 mA cm-2) and the logarithm base 10 of the ionic strength of the electrolyte, NaCl and Na2SO4 (&micro; = 0.05, 0.15 and 0.5 mol L-1), in order to study the effect of current density, concentration and nature of the electrolyte to determine the best diethyl phthalate degradation condition. The monitoring of DEP concentration was made by high-performance liquid chromatography (HPLC) and the mineralization was accompanied by total organic carbon (TOC) analysis. They obtained higher percentages of removal and mineralization with the use of higher current densities and in the presence of high concentrations of NaCl in comparison with Na2SO4. Thus, there was obtained the removal of 63.2 % and 63.9 % mineralization in 0.5 mol L-1 NaCl and current density of 40 mA cm-2, whereas for Na2SO4 (&micro; = 0.5 mol L-1) and 40 mA cm-2 was removed 51.3 % and 53.0% mineralized DEP. The DEP degradation mechanism was determined in NaCl and Na2SO4, by HPLC-MS under the conditions mentioned above, identifying the molecular ion of m/z 149 and 177 on both electrolytes, corresponding to protonated phthalic anhydride and an adduct of phthalic anhydride with C(2)H(5)(+), respectively, characteristic ions of DEP fragmentation, besides the ion m/z 239 over Na2SO4 corresponding to diethyl 3-hydroxyphthalate. The DEP degradation takes place through the aliphatic chain.